protein-protein interactions masoud youssefi, md,phd division of microbiology/virology
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Protein-protein interactions
Masoud Youssefi, MD,PhDDivision of microbiology/virology
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Introduction• important field in cell biology, biochemistry Localization and trafficking posttranslational modifications signaling networks • also important field in viral replication• very difficult to predict• two main patterns: ■ domain-domain interactions ■domain-peptide interactions
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An example: virion assembly
• The components come together and the Nucleocapsid is formed which in turn will become completed to the whole particle.
• The assembly process begins when concentration of structural proteins is enough within the cell to drive the process.
• Many protein-protein, protein-nucleic acid and in case of membrane viruses protein-membrane (fatty acid) interactions are needed.
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The mechanism of interaction
• Non-covalent so reversible• Van del waals forces• Hydrophobic interactions• Electrostatic bonds• Hydrogen bonds• For strong couplings very accurate force field
potentials are needed
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How to study protein protein interaction?
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Overview of techniques
• Gel filtration• Far western blot• Affinity
chromatography• Co-
immunopercipitation• Capillary elecrophoresis• Biosensor
• FRET microscopy• Confocal microscopy• 2 hybrid assay• Protein microarry• Maspec• NMR• Co-crystallization for
crystallography
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Gel filtration chromatography
• Also called ”Size exclusion”• Porous made up of cross-
linked polymers• Small molecules are
trapped by the beads• For self assembling proteins
monomers come later
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Far western blot• Also called ”Blot overlay”• Fractionating proteins on
SDS-PAGE• Blotting to nitocellulose or
PVDF membrane• Overlaying with a solution
of the protein of interest• Binding the added protein
to an immobilized protein on the membrane
• Detection with antibody against the overlaying protein
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Co-Immunoprecipitation• Protein A binds to
antibodies• Sepharose beads coated
with protein A• Specific antibody binds to
the protein of interest • The complex is precipitated
by binding to the beads via protein A
• Proteins are released from beads by boiling
• Western blot
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Affinity chromatography
• In the case of His- tagged proteins• The His-tagged protein binds to nickel or
cobalt column • His-tagged protein and it’s associated protein
are eluted from the column by adding imidazole
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Fluorescence resonance energy transfer (FRET)
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FRET cont• Cyan fluorescence protein (CFP) and yellow
fluorescence protein (YFP) are spectral variants of GFP
• Plasmid constructs to fuse the proteins of interest to CFP and YFP
• Co-transfection of plasmids to the cells• Fixation of the cells and view by confocal microscopy• Disadvantage:False negative results: If the fluorophores are over 200Ǻ apart while the
proteins interact with each other, no signal will be observed
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FRET using CFP & YFP
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Fluorescence resonance energy transfer (FRET)
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Yeast two-hybrid assay
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Yeast two hybrid assay
• Transcription factor, Gal4p, has DNA binding (BD)(aa1-147) and transcriptional activator(AD)(aa768-881) domains
• Stimulates transcription at a promoter reconized by Gal4p (upstream activating sequence,UAS)
• Lac Z reporter gene encodes beta-galactosidase which produces blue pigment when the colony is grown in a media containing X-Gal
• Disadvantage:time consuming!
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2 Hybrid system
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Mamalian two-hybrid assay• Is analogous to Y2H assay• Plasmids: 1)Gal4pBD-fusion vector 2)VP16AD-fusion vector(viral activator) 3)luciferase reporter plasmid contaning multiple copies of Gal4p binding sites(UAS)
• Co-transfection: in the case of interaction, luciferase activity will be detected
• Advantage: good for studying mammalian proteins: they may not fold correctly in yeast or they may require post-tranlational modifications for protein interaction
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what are biosensors?
• Transducer converts physical change(heat, change in charge, light absorbance, mass) into an electrical signal
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Confocal microscopy
• A good technique to detect intracellular co-localization of proteins
• Point scan laser system minimizes overlaps in image (perfect for imaging Co-localization of proteins)
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Confocal microscopy cont.
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Overview of techniques
• Gel filtration• Far western blot• Affinity
chromatography• Co-
immunopercipitation• Capillary elecrophoresis
• FRET microscopy• Confocal microscopy• 2 hybrid assay• Maspec• NMR• Co-crystallization for
crystallography
![Page 23: Protein-protein interactions Masoud Youssefi, MD,PhD Division of microbiology/virology](https://reader036.vdocument.in/reader036/viewer/2022062421/56649d615503460f94a42da1/html5/thumbnails/23.jpg)
• Thank you!